GDAP1 mutations in Italian axonal Charcot-Marie-Tooth patients: Phenotypic features and clinical course.

Mutations in the ganglioside-induced differentiation associated-protein 1 (GDAP1) gene have been associated with both autosomal recessive (AR) and dominant (AD) Charcot-Marie-Tooth (CMT) axonal neuropathy. The relative frequency of heterozygous, dominant mutations in Italian CMT is unknown. We investigated the frequency of dominant mutations in GDAP1 in a cohort of 109 axonal Italian patients by sequencing genomic DNA and search for copy number variations. We also explored correlations with clinical features. All cases had already been tested for variants in common axonal AD genes. Eight patients (7.3%) harbored five already reported heterozygous mutations in GDAP1 (p.Arg120Gly, p.Arg120Trp, p.His123Arg, p.Gln218Glu, p.Arg226Ser). Mutations had different penetrances in the families; the onset of symptoms is in the first decade and progression is slower than usually seen in GDAP1-related AR-CMT. We show that the relative frequency of mutations in GDAP was slightly higher than those observed in MFN2 and MPZ (7.3% vs 6.3% and 5.0%). The relatively milder clinical features and the quite indolent course observed are relevant for prognostic assessment. On the basis of our experience and the data reported here, we suggest GDAP1 as the first gene that should be analysed in Italian patients affected by CMT2.

Process optimization and biocompatibility of cell carriers suitable for automated magnetic manipulation.

There is increasing demand for automated cell reprogramming in the fields of cell biology, biotechnology and the biomedical sciences. Microfluidic-based platforms that provide unattended manipulation of adherent cells promise to be an appropriate basis for cell manipulation. In this study we developed a magnetically driven cell carrier to serve as a vehicle within an in vitro environment. To elucidate the impact of the carrier on cells, biocompatibility was estimated using the human adenocarcinoma cell line Caco-2. Besides evaluation of the quality of the magnetic carriers by field emission scanning electron microscopy, the rate of adherence, proliferation and differentiation of Caco-2 cells grown on the carriers was quantified. Moreover, the morphology of the cells was monitored by immunofluorescent staining. Early generations of the cell carrier suffered from release of cytotoxic nickel from the magnetic cushion. Biocompatibility was achieved by complete encapsulation of the nickel bulk within galvanic gold. The insulation process had to be developed stepwise and was controlled by parallel monitoring of the cell viability. The final carrier generation proved to be a proper support for cell manipulation, allowing proliferation of Caco-2 cells equal to that on glass or polystyrene as a reference for up to 10 days. Functional differentiation was enhanced by more than 30% compared with the reference. A flat, ferromagnetic and fully biocompatible carrier for cell manipulation was developed for application in microfluidic systems. Beyond that, this study offers advice for the development of magnetic cell carriers and the estimation of their biocompatibility.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

OBJECTIVE: To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. METHODS: Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). RESULTS: At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. CONCLUSIONS: Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

[Simultaneous ultrasonography and arthroscopy for the study of the joint environment: indications and limits]. / Ecografia e artroscopia in contemporanea per lo studio dell'ambiente articolare: indicazioni e limiti.

Arthroscopy is a mini-invasive technique that allows the direct observation of the joint cavity and the execution of diagnostic and therapeutic procedures; arthroscopy needs a very long learning-time curve as well as dedicated spaces and instruments. Ultrasonography is an imaging technique that enables to perform an immediate extension of the standard physical examination. The opportunity to visualize soft tissues, to obtain multiplanar and dynamic images in real time makes this practice easy repeatable at low costs. Ultrasonography allows to detect a variety of changes during inflammatory processes. The wide experience in arthroscopy of rheumatic patients acquired through the years by our team at the G. Pini Institute led us to study in vivo, during arthroscopy, the correspondence between arthroscopic and ultrasonographic images. Up to now three knee arthroscopies have been conducted with the double equipment (ultrasonographic and arthroscopic devices) in operating room. In our experience, the combination of the two methods in operating room may improve the validation of ultrasonography with arthroscopy as gold standard, helps to train the ultrasonographer to give immediate answers in order to clear the doubts aroused by ultrasonographic images; it also allows the arthroscopist to visualize the deeper layers of the synovial membrane making double guided targeted biopsies possible. Limits are the complexity of the procedure (instruments, operators, spaces, training of the doctors), the loose of power-doppler signal with the blood tourniquet and the always difficult evaluation of cartilage.

An amperometric glucose biosensor prototype fabricated by thermal inkjet printing.

The prototype of an amperometric glucose biosensor was realized by thermal inkjet printing using biological and electronic water-based inks, containing a glucose oxidase (GOD) from Aspergillus niger and the conducting polymer blend poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS), respectively. The biosensor was fabricated microdepositing PEDOT/PSS and GOD, in sequence, on ITO-glass, by a commercial inkjet printer, with the help of a commercial software. High density microdots matrices were so-realized, with a calculated resolution of about 221 x 221 dpi (dot per inch). By means of a rapid and easy assay it was demonstrated that no activity loss occurred upon the printing of GOD, despite of the use of a thermal printhead. The device was encapsulated in a semipermeable membrane of cellulose acetate, applied by dip-coating, in order to prevent dissolution of the enzyme and/or PEDOT/PSS in water. The preliminary response of the electrode was measured in an aqueous glucose solution in the presence of ferrocenemethanol (FeMeOH) as a mediator, and resulted linear up to 60 mM in glucose. The best sensitivity value achieved was 6.43 microAM(-1) cm(-2) (447 nAM(-1) U(-1) cm(-2)). The characteristics of the device, and the possible performance improvements have been analyzed and discussed. The reported findings indicate that inkjet printing could be a viable instrument for the easy construction of a working biosensor via direct digital design using biological and conductive polymer based inks. Such an approach may be seen as an example of "biopolytronics".

Treatment of malignant gliomas: a new approach.

The aim of this study is to describe the authors' experience with intra-arterial ACNU chemotherapy of malignant gliomas. The prognosis of cerebral malignant gliomas remains poor, whatever traditional therapy is applied. ACNU is a well tolerated nitrosourea with a strong antimitotic effect on neurogenic cells both in vitro and in vivo; this drug has enhanced efficacy when used at high concentrations, particularly as an intraarterial infusion. Seventy-six patients have been studied to date, 68 of whom are evaluable; these patients were treated by intra-arterial infusion of ACNU (100 mg/m2) every 6 weeks, with a mean of 2.5 courses per patient. The objective response (OR) was 28% and analysis of pretreatment factors revealed that survival was influenced by histological grade, other types of therapy applied, and age. In general IAC is well tolerated and the response and survival appear to be better than with systemic chemotherapy.

Transcranial Doppler sonographic monitoring during cerebral aneurysm embolization: a preliminary report.

BACKGROUND AND PURPOSE: The wide application of embolization in the treatment of aneurysms has created the need for an intraprocedural means to anticipate a poor outcome by monitoring hemodynamic changes in the brain. METHODS: Transcranial Doppler sonography was used to monitor flow velocity in the middle cerebral artery (MCA) in 23 patients undergoing embolization with Guglielmi detachable coils (GDCs) of either incidental or symptomatic intracranial aneurysms. Sonographic values were recorded from the ipsilateral MCA at the beginning, middle, and end of the interventional procedure and 24 hours afterward. RESULTS: No complications occurred in 15 patients. In these cases, sonography showed an average decrease in MCA flow velocity of 2.7% after GDC application, returning to baseline at the end of treatment and then increasing by about 17% 24 hours later. In four patients with vasospasm on posttreatment angiograms, MCA flow velocity increased to values higher than 120 cm/s after GDC application, returning to baseline after 24 hours. In four patients with ischemic complications (two transient ischemic attacks, one stroke, one vascular death), MCA flow velocity decreased more than 30% and did not return to preoperative values within 24 hours. CONCLUSION: The application of transcranial Doppler sonographic monitoring during endovascular treatment may help to identify patients at risk for posttreatment cerebral ischemia.